In 2012, we made progress in the following projects: TCR signal transduction in response to partial agonists, Fos induction in T cells, feedback between calcium signals and Map kinase signals downstream of TCR stimulation and its influence on cFos induction, cross talk among common gamma chain family of cytokine receptors in T cells and role of RhoH in TCR signaling. Additionally in collaboration with the labs of Jon Yewdell and Jack Bennink we demonstrated peptide specific clustering of MHC class I molecules at the surface of virally infected antigen presenting cells. Due to the diverse nature of TCR and peptide loaded MHC complexes, TCRs from different cells bind their cognate peptide-MHC complexes (MHCp) with varying affinities. To understand signaling downstream of TCR in response to ligands of varying affinity we employ the model system consisting of T cells from a TCR transgenic mouse (AND) stimulated with altered peptide ligands. As the potency of peptides is reduced, the cells ability to cause calcium signaling is compromised while activation of MAP kinases is preserved. Upon binding peptide MHCp, TCRs undergo micron scale clustering in the plasma membrane called TCR microclusters. TCR microclusters act as a biochemical unit associated with proximal TCR signaling where various signaling molecules are recruited. We have visualized the recruitment of GFP tagged LAT, Zap70 and Grb2 transfected in in-vitro activated AND TCR transgenic T cells using Total Internal Reflection Fluorescence microscopy (TIRFM) of cells interacting with glass supported lipid bilayers containing lipid anchored peptide-MHC complexes, ICAM-1 and CD80. We find as expected that high potency ligands recruit Zap70, Lat and Grb2 to the microclusters. One of the medium potency ligands causes the phosphorylation and recruitment of Lat to the microclusters without the recruitment of Zap70. We are exploring the role of other kinases that maybe responsible for the phosphorylation of Lat. One of the ligands we have studied leads to the generation of Erk signals in the absence of calcium signals. These Erk signals are correlated with the specific recruitment of Grb2 to the TCR microclusters in the absence of Lat recruitment. Our results have demonstrated a qualitative difference in TCR signaling in response to MHCp of varying affinity. Immediate early genes such as cFos and Egr-1 are dependent on calcium and MAP kinase signaling via, CRE (cyclic AMP response element) and SRE (serum response element) in their promoters respectively. We have explored feedback between calcium and MAP kinase signaling and found that upon inhibiting calcium signals in T cells, Erk responses are increased and JNK and p38 responses are diminished. On the other hand, inhibiting Erk caused decreased calcium signals while, inhibiting p38 caused increased calcium signals. We have further studied the influence of calcium and MAP kinase signaling in the induction of immediate early genes cFos and Egr-1. We find that both these genes are absolutely dependent on calcium signaling for their induction and are only partially dependent on MAP kinases. Their calcium dependence is exerted via CREB phosphorylation which is calcium dependent. Additionally, dominant negative CREB inhibits cFos induction. The common gamma chain family of cytokine receptors (IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21) shares the gamma chain for signaling. Since many receptors are expressed on T cells at the same time, it is not clear how these receptors share the gamma chain and if it is ever limiting for signaling. We have quantified the numbers of each of the members of this family on the surface of nave polyclonal T cells and find that a simple model where each of these receptors are paired with the gamma chain is not possible as the gamma chain is not abundant enough. Intriguingly, dose response curves show that the STAT phosphorylation is saturated at receptor occupancies of less than 1 per cent, suggesting that only a small fraction of receptors need to be pre-associated with the gamma chain for full signaling. We further found that IL-7 is able to inhibit signaling in response to IL-4 and IL-21 when cells are sequentially stimulated with IL-7 and IL-4 or IL-21. We are exploring the biochemical mechanisms behind this competition as it is not a straightforward competition for gamma chains. RhoH is a member of the Rho family of GTPases, but is unusual in that it does not have GTPase activity, and is constitutively GTP bound. RhoH is indispensable for TCR signaling as RhoH deficient mice have defective thymocyte development. RhoH is regulated by tyrosine phosphorylation and is thought to regulate the membrane localization of Lck and Zap70. We have found that RhoH localizes to TCR microclusters in a signal strength dependent manner. We have found that mutants of RhoH that cannot be phosphorylated have altered amounts in the cSMAC of immune synapses. These experiments will allow us to understand how RhoH regulates the concentration of Zap70 and Lck in TCR microclusters. We have an extensive on going collaboration with the lab of Martin Meier-Schellersheim, wherein we are building computational models of signaling via the common gamma chain family of cytokine receptors and the role of RhoH in TCR signaling. This collaboration will help us refine our experiments and the experimental results will in turn refine the computational models. In collaboration with the labs of Jon Yewdell and Jack Bennink we have studied the clustering of MHC class I proteins on the surface of antigen presenting cells using total internal reflection fluorescence microscopy. The clustering of MHC class I molecules has been known for a while. We, however, found that clusters of MHC class I were peptide specific when source antigens were presented by vaccinia or vesicular stomatis virus. The intracellular distribution of these peptide loaded MHC molecules were also distinct, suggesting that the cells had a mechanism of sorting MHC molecules based on the peptide presented and this sorting dependent on the cytoplasmic domain of MHC class I molecules. This work is now in press. In collaboration with the lab of Stephen Lockett at NCI, we have set up a new microscope to perform Fluorescence Correlation Spectroscopy. This technique will help us monitor molecular interactions in the plasma membrane and determine the diffusion coefficient of proteins in solution and in the plasma membrane. We can also monitor the fluctuations in FRET signals at time resolution of a few microseconds on this microscope. This will allow us to measure calcium transients in cells as well as measure conformation changes in proteins. We published a video article describing a method of transfecting T cells and imaging them using TIRF microscopy. The article has a detailed description of setting up a two channel simultaneous acquisition TIRF microscope and has been helpful for a lab at MIT where they are in the process of replicating the system. We also wrote a book chapter describing different fluorescence techniques to study events in the plasma membrane. In the near future we shall be submitting three manuscripts covering the three projects described.